Since the advent of HAART therapy for HIV, co-infection with hepatitis C virus (HCV) has emerged as a major health problem. HIV/HCV co-infected patients progress more rapidly towards end-stage liver disease than HCV mono-infected patients, and co-infected patients present special challenges given the hepatotoxicity of many HIV therapeutics. Current treatment for HCV consists of a combination of pegylated interferon alpha and the nucleoside analog ribavirin. In HCV genotype 1 infected patients (the most common HCV genotype in the US), this therapy clears infection in less that 50% of those treated. The goal of this proposal is to identify and use small molecules to learn more about the biology of HCV replication. In collaboration with the Rockefeller University High- Throughput Screening facility, chemically diverse small molecule libraries will be screened for anti- HCV activity using a cell-based replicon assay. Inhibitory, non-cytotoxic compounds will be counter-screened against other members of the Flaviviridae family, including bovine viral diarrhea virus and yellow fever virus. While compounds with pan-flavivirus activity are of interest, we will initially focus on those specific for HCV. Compounds will be tested in cell-based and cell-free assays to probe the step(s) in HCV replication that are inhibited. In parallel, inhibitory compounds will be used to screen for resistant mutants and to determine if resistance maps to a specific viral protein or RNA element. Future work will include biochemical and structural studies aimed at understanding the mechanism of action of selected HCV inhibitors. These studies will provide useful chemical probes for dissecting different steps in the HCV replication cycle with the hope of identifying novel targets for drug development.